Measuring TEER in X-ray-Irradiated Epithelial Cells Co-cultured With PBMCs

Samenvatting

Source: Babini, G., et al. A Co-culture Method to Investigate the Crosstalk Between X-ray Irradiated Caco-2 Cells and PBMC. J. Vis. Exp. (2018)

This video demonstrates the measurement of trans-epithelial electrical resistance in irradiated Caco-2 cells, both in the presence and absence of PBMC cells. This method enables the study of the synergistic effects of radiation and immune cells on the epithelial cell barrier.

Protocol

1. Irradiation Set-up

Note: Irradiation of Caco-2 cells was performed at the radiotherapy department of Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) S. Maugeri (Pavia, Italy) with a linear accelerator routinely used to treat different types of cancers.

1. Set photon X-ray energy to 6 MV peak. The linear accelerator operates in a pulsing regime (time between two consecutive pulses approximately of 4 ms; duration of a single pulse about 5 µs with a dose rate up to 1 × 10^-3 Gy/p).
2. Place the 6-well plates containing inserts with Caco-2 on the trajectory of X-rays on a 1.4 cm-thick Plexiglas sheet (corresponding to a distance slightly greater than the build-up of the used radiation) at 100 cm from the source of radiation.
3. Place a 0.5 cm-thick bolus on each sample before the irradiation occurs to guarantee the backscattered radiation component and the charged particles' equilibrium.
4. Irradiate the cells (doses in the range 2 - 10 Gy) with a flat and symmetric (± 2%) 20 x 20 cm₂ radiation field and a dose-rate of 3 Gy/min.
   NOTE: Control "sham"-irradiated cells underwent the same procedural conditions of the irradiated ones, without entering the irradiation room (received dose: 0 Gy).
   CAUTION: Ionizing radiation-producing devices must be used only by specialized personnel.

2. Trans-Epithelial Electrical Resistance (TEER)

1. Seed 5 × 10⁵ Caco-2 cells one week prior to irradiation in 6-well plate co-culture inserts (polyethylene terephthalate (PET), 2 x 10⁶ pores/cm2).
2. 1 h before irradiation, measure the TEER with a voltmeter/ohmmeter.
3. Irradiate cells as described in steps 1.1 - 1.4.
4. Incubate Caco-2 cells in the presence/absence of co-culture with PBMC at 37 °C in a humidified atmosphere containing 5% CO₂.
5. For the first few hours post-irradiation, measure TEER every hour, and subsequently every 3 hours up to 48 hours.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
ThinCert 6 Well Cell Culture Inserts for Multiwell Plates	Greiner Bio-one	657610	Equipment
Cell Culture Multiwell Plate, 6 Well	Greiner Bio-one	657160	Plastic
Cell Culture Multiwell Plate, 24 Well	Greiner Bio-one	662160	Plastic
RPMI 1640 without L-Glutamine	Lonza	12-167F	Reagents
Millicell-ERS voltmeter/ohmeter	Millipore	MERS 00001	Equipment